Towards our goal of understanding DNA transposition by the IS608 transposase, TnpA, we have recently reconstituted the entire recombination reaction in vitro using single-stranded DNA, and have solved the structures of six different complexes of TnpA with various oligonucleotides representing sequences located at the transposon ends. These structures have provided detailed views of sequential steps along the recombination pathway including (i) the pairing of transposon ends caused by TnpA binding to DNA hairpin structures located close to each end; (ii) the large-scale conformational change induced by hairpin binding that causes the transition from an inactive enzyme to one with a correctly assembled active site;(iii) the orientation of the transposon ends in the active site;and (iv) the mode of target site recognition. This last observation is particularly important as it immediately suggests ways to modify the system so that alternate targets might be used for transposition. In particular, we hope to devise methods to allow targets longer than a tetranucleotide to be used. If we can do this, this might allow the precise introduction of exogenous genes into benign locations in chromosomes or places where gene expression can be appropriately controlled in a cell- and development-specific manner. We are also investigating another member of the IS200 superfamily, ISDra2, to determine if features of IS608 transposition might be generalizable to the entire superfamily. ISDra2 is particularly interesting as its transposition is specifically induced in Deinococcus radiodurans upon UV or gamma irradiation (Mennecier et al., 2006). We have solved a series of Dra2-DNA complexes, and shown that although many of the principles of targeting found for IS608 are also applicable, there are subtle yet mechanistically important differences. One noteworthy difference is that ISDra2 recognizes five nucleotides at its target site, rather than the four of IS608. The manner in which ISDra2 accomplishes this extends our understanding of the mechanism of transposition. We have also been able to structurally capture the pre-cleavage state along the pathway, a snapshot that eluded us with the IS608 system. We have been able to demonstrate that transposition by IS608 can indeed be redirected to altered target sites both in vitro and in vivo. The rationale for this was provided by our structural results that showed that target site recognition was accomplished by base pairing interactions between the target site and an internal segment of transposon DNA. Therefore, changing this internal segment should yield a transposon that would target a new and predictable target site. Our experimental results are consistent with this prediction. Currently we are conducting experiments to determine whether target site specificity can be extended beyond the naturally occurring tetranucleotide sequence. Obviously, if successful, such a transposition system could be very useful for a variety of genomic applications. Curcio, M.J. and Derbyshire, K.M. (2003) Nat. Rev. Mol. Cell. Biol. 4, 865-877. Debets-Ossenkopp, Y.J., et al. (1999) Antimicrob. Agents Chemother. 43, 2657-2662. Kersulyte, D., et al. (2002) J. Bacteriol. 184, 992-1002. Mennecier, S., Servant, P., Coste, G., Bailone, A., and Sommer, S. (2006) Mol. Microbiol. 59, 317-325. Sebaihia, M. et al. (2006) Nature Genet. 38, 779-786.